Project Summary: Nucleosomes are the fundamental and repeating units of chromatin, consisting of DNA wrapped around a histone octamer. Alterations in chromatin structure and function dramatically impact downstream gene expression and cellular physiology. This epigenetic regulation is controlled by two major modifications: histone post-translational modifications (PTMs; such as histone methylation, acetylation, and ubiquitination) and DNA methylation. Significantly, specific alterations in the epigenetic landscape are associated with a diverse array of human diseases most notable of which are cancers. Understanding this so-called ?histone code? is essential for developing targeted therapies that manipulate or alter epigenetic signaling to treat human disease. As a key step toward deciphering the histone code, EpiCypher? Inc. has initiated a robust research program to develop recombinant nucleosome-based reagents and assays, which capitalize on physiological properties of chromatin architecture. Recent studies show that histone and DNA methylation signaling pathways are interdependent and may synergistically alter the activity of chromatin modifying enzymes. These data not only support the hypothesis that histone- and DNA methylation-dependent signaling are mechanistically linked, but provide a unique opportunity for novel drug discovery. Currently, there are no commercially available research tools to biochemically profile the interplay between specific histone PTMs and DNA methylation on a single assay substrate. Here, we will develop an innovative nucleosome-based assay platform engineered to leverage the crosstalk between histone PTMs, DNA methylation, and chromatin-interacting/modifying enzymes to accelerate drug discovery. In Phase I, we will develop methods to generate recombinant nucleosomes carrying unique DNA and histone methylation profiles. Using these reagents as biochemical substrates, we will demonstrate feasibility that this assay platform will be useful for drug discovery by establishing novel effector- binding and enzymatic assays using proteins known to interact with DNA and/or histone methylation, such as UHRF1. UHRF1 is an E3 ubiquitin-protein ligase whose activity is greatly enhanced in the presence of hemimethylated DNA and histone H3K9me2. Significantly, UHRF1 overactivation is associated with bladder and colon cancer. In Phase 2, we will continue to optimize the commercial synthesis of designer nucleosomes assembled using methylated DNA templates, which we will use to develop additional novel effector binding and enzymatic assays. These reagents and assays will be assembled into a series of stand-alone kits for innovative drug discovery. In addition, we will develop a designer nucleosome-based UHRF1 inhibitor screen that leverages the molecular interactions between UHRF1 and DNA/histone methylation to identify context- specific inhibitors of UHRF1 for cancer therapeutic development. The breakthrough technology developed here is greatly needed by the epigenetic research community and will enable the identification of novel drug targets as well as the development of precision therapeutics.